氧化氮对抗脑缺血再灌注所致脑细胞凋亡的机制探讨

目的探讨一氧化氮（nitric oxide，NO）对局灶性脑缺血再灌注所致神经细胞损伤的影响及影响机制。方法将SD大鼠随机分为假手术组（N组）、脑缺血再灌注组（MCAO组）、脑缺血再灌注加侧脑室微量注射20mmol／L的L-Arg5肚组（L-Arg组）及脑缺血再灌注加侧脑室微量注射20mmol／L的L-NAME5 μl组（L-NAME组），作脑缺血30min，再灌注12h、24h和2d，冰冻切片，相邻切片分别作焦油紫染色、NOS免疫组化、NOSmRNA原位杂交、TUNEL法原位检测凋亡细胞。结果N组NOS的活性弱阳性表达；MCAO组术后24hNOS的表达明显增强，与各组比较，P〈0．05；L-Arg组术后12h小血管内皮细胞出现NOS的阳性高表达，术后24h神经细胞NOS的阳性表达最高，与各组比较，P〈0．05；L-NAME组各时间点NOS活性的表达为阴性或可疑阳性，与各组比较P〈0．05，NOS的活性明显受到抑制。凋亡细胞的计数结果为N组26．3±4、2个，MCAO组62±4．2个，L-Arg组40、6±2．7个，L-NAME组78．3±3．3个，P〈0．05。结论适量NO可有效降低细胞凋亡的发生，减轻脑缺血再灌注所致的神经细胞的损伤。

The Effect of Nitric Oxide on Antagonizing Brain Cell Apoptosis Caused by Cerebral Ischemic Reperfusion

Objective To investigate the influence of NO on cell damage in rat neurons after focal cerebral ischemia reperfusion. Methods Cerebral ischemia for 30 minutes was performed in SD rats. The rats were randomly divided into 4 groups ： （1） control group, （2） sham-operation group, receiving injection of 5 kd NS in the lateral cerebral ventricle, （3） DArg group, with a microinjection of 5 μl 20 ramol/L DArg into the lateral cerebral ventricle, and （4） DNAME group, with a microinjection of 5 μl 20 mmol/L L-NAME into the lateral cerebral ventricle. Serial frozen sections were made from tissue samples taken at reperfusion 12 hours,24 hours and 2 days after cerebral ischemia for 30 minutes. Cresyl violet staining, NOS immunohistochemistry, NOS mRNA in-situ hybridization and TUNNEL were done to detect apoptotic cells in histological sections. Results Weakly NOS signal in the control group was detected. However,the expression of NOS in MCAO group at 24 hours after operation was more stronger than that in other groups （ P 〈 0.05）. At 12 hours after operation, NOS in endothelial cells of small blood vessels in L-Arg-treated rats was highly increased and NOS in neurons at 24 hours after operation was highest （ P 〈 0.05）. The expression of NOS in L-NAME group was negative or weakly positive （P 〈 0.05）. The activity of NOS was inhibited obviously. Apoptotic cells in the control, MACO,L-Arg and L-NAME groups were 26.3 ± 4.2,62 ± 4.2,40.6 ± 2.7 and 78.3 ± 3.3, respectively （ P 〈 0.05）. Conclusion Nitric oxide can effectively inhibite cell apoptosis and alleviate the damage of neurons after focal cerebral ischemia reperfusion.